Parkinson's disease (PD) is characterized by progressive and selective loss of dopaminergic neurons in substantia nigra pars compacta. Emerging evidence suggests that the degeneration of dopaminergic neurons in PD is mediated via mitochondrial dysfunction and subsequent activation of various apoptosis-execution cascades. Thus, the long-term goal of this proposal is to identify novel neuroprotective agents that are capable of blocking the mitochondrial apoptosis-signaling pathway in dopaminergic neurons induced by Parkinsonian neurotoxins. HSP27 is a member of the small heat shock protein family, a group of ubiquitous stress proteins that are expressed in virtually all organisms. The expression of HSP27 is induced in the brain after several forms of injury, and its neuroprotective role has been strongly implicated. In addition to its knovm function as a protein chaperone, recent studies suggest that HSP27 has potent anti-apoptotic effects, and it appears that HSP27 gains its anti-apoptotic properties after undergoing serine-specific phosphorylation. We have recently created transgenic mice overexpressing either the wild-type HSP27 or a nonphosphorylatable HSP27 mutant. Using both transgenic and gene-transfection approaches, we have obtained exciting preliminary results which suggest that overexpression of HSP27 protects against dopaminergic cell death induced by PD-relevant insults in both in vivo and in vitro settings, that the neuroprotective effect of HSP27 is dependent on phosphorylation-mediated activation of the protein, and that HSP27 may achieve the neuroprotective effect via inhibiting ASKi/JNK-mediated mitochondrial-apoptosis signaling pathway. This proposal attempts to test the hypothesis that enhanced expression and phosphorylationdependent activation of HSP27 protects against dopaminergic cell death via novel antiapoptotic mechanisms. The following specific objectives are proposed: Aim 1. Test the hypothesis that transgenic overexpression and phosphorylation-dependent activation of HSP27 protects against dopaminergic cell death in the brain following Parkinsonian neurotoxins. Aim 2. Test the hypothesis that the neuroprotective effect of HSP27 against dopaminergic cell death is mediated via novel anti-apoptotic mechanisms involving the disruption of ASKi/JNK-dependent mitochondrial signaling pathways. Aim 3. Test the hypothesis that TAT protein transduction domain-mediated or adeno-associated virus (AAV)-mediated delivery of HSP27 into the brain protects against dopaminergic cell death induced by Parkinsonian neurotoxins. Studies in both in vivo and in vitro models are proposed. The in vivo animal models of 6-OHDA and rotenone neurotoxicity mimics important aspects of pathophysiological changes in relevance to PD. The in vitro models employing various Parkinsonian neurotoxins (6-OHDA, MPP-t-, rotenone) will complement the in vivo studies by allowing for precise mechanistic studies to be performed.